In humans, a single enzyme, alkyladenine DNA glycosylase (AAG), safeguards the cell against a wide variety of alkylated and deaminated DNA bases. As DNA alkylating agents are often used in the treatment of cancer, the molecular mechanism of AAG action is a necessary step, not only toward understanding the biology of base-excision repair, but also toward developing more effective cancer treatments. To understand how AAG recognizes a large subset of DNA adducts, I will define the kinetic and thermodynamic parameters for the interaction of AAG with damaged DNA substrates. These experiments will identify individual steps in the AAG reaction at which specificity is expressed and address the central question of how AAG discriminates between normal bases and damaged bases. These efforts will be complemented by high resolution crystallographic experiments to define the structural basis of specificity. The framework for AAG action on defined DNA substrates in vitro will be extended to include other purified DNA repair components, such as human AP-endonuclease and HR23 proteins, to evaluate the molecular nature and biological significance of interactions between AAG and other DNA repair proteins. Experiments with long, defined DNA substrates will test whether AAG scans DNA in a processive fashion. If AAG does scan DNA via linear diffusion, I will characterize the structural features of AAG and the DNA itself that are required for efficient scanning, to elucidate how DNA lesions are located by AAG.